The peripheral nervous system has retained a remarkable capacity for axonal regeneration. In response to injury, well-characterized neuron intrinsic signaling pathways mount a regenerative response that eventually leads to spouting of axonal growth cones. Promoted by well-defined growth factors, growth cones extend along denervated Schwann cells that they utilize as a general regeneration pathway, yet at branch points individual regenerating axons have to select the correct path towards their original targets. Although accurate regeneration of axons to their original targets is critical for the functional recovery, the cellular and molecular mechanisms by which regenerating axons select their original targets are not well understood. We recently established an in vivo system to monitor and quantify target selective re-innervation in live intact animals. Using this system we discovered that following transection of the dorsal and ventral motor nerve branch, regenerating zebrafish motor axons exhibit a strong preference for their original muscle territory, providing compelling evidence for the existence of molecular mechanisms for target-selective regeneration. To identify the genes underlying this process we surveyed mutants in genes with known roles in neural development. We identified four genes that do not promote axonal regrowth per se, but rather provide target selectivity to regenerating axons. The experiments in this proposal build upon the findings that in mutants for the robo2 guidance receptor and for the exostosin like 3 (extl3) glycosyltranferase motor axons develop normal but regenerating dorsal nerve axons frequently select incorrect, ectopic trajectories, invading lateral and ventral territories. While both genes play well-defined roles in neural development, their function in regeneration is not understood. The experiments in this proposal will define the mechanisms by which these two genes promote target selective regeneration. In Aim 1 we will determine the molecular mechanisms through which robo2 functions in regeneration, e.g. as an axonal Slit receptor, or alternatively as Schwann cell receptor. In Aim 2 we will determine the cellular mechanisms by which robo2 guides dorsal nerve regeneration, e.g. by correcting pathfinding mistakes at the choice point, and/or by directing pioneering axons towards their original path, thereby providing a regeneration pathways for follower axons. Finally, in Aim 3 we will determine whether exostosin like 3 (extl3) guides regenerating axons through its role in heparan sulfate production or via its unique surface receptor domain. Combined, the proposed studies will make significant contributions to the fundamental science of how transected axons return to their original targets. This will results in a better understanding of peripheral nerve regeneration across the board and will help to address the urgent therapeutic needs for patients suffering from peripheral neuropathies caused by diabetes, injury, and autoimmune disorders.